molecular cloning
How to read a plasmid map
A decision procedure for reading origin class, resistance marker, promoter arrow, MCS, tags and cutters on a plasmid map before you order an enzyme.
8 October 2026 · 11 min read
Topic cluster
Molecular cloning
Plasmid maps, assembly methods, colony screening and the evidence that an insert is present, oriented and intact.
All topicsBuffers and laboratory water26Cell culture26Chromatography26Cold chain and sample storage26Cytometry and microscopy26DNA sequencing26Gels and blotting26Gene expression26Genome editing26Immunoassays26Laboratory labware26Laboratory safety26Liquid handling26Microbiology26Molecular cloning26Nucleic acid purification26PCR and amplification26Protein biochemistry26Proteomics26Research sourcing and study design26
biosafetyspecificationsequencingglossarytroubleshootingcloningcontrolsexpressionsangertransformationcoloniesligationglycerol stockplasmid mapscreeningselectioncodonscolony pcr
26 articles
molecular cloning
Miniprep yield and downstream sequencing
Decide whether a miniprep is clean enough to sequence or should be repeated, using copy number, culture volume, and carryover rather than a promised yield.
8 October 2026 · 7 min read
molecular cloning
Orientation of an insert in an expression vector
Why a backward insert still grows on antibiotic, and how an asymmetric digest, a junction primer or a sequence read shows which way the ORF points.
8 October 2026 · 8 min read
molecular cloning
Overlap assembly design rules
How to troubleshoot Gibson-style overlaps of about 15 to 40 bases when repeats, a melting mismatch, a short insert or exonuclease chew-back wrecks the join.
8 October 2026 · 8 min read
molecular cloning
Phosphatase treatment and self-ligation
When to strip 5-prime phosphates from a cut vector so it cannot recircularise, why the insert must keep its phosphates, and how leftover background looks.
8 October 2026 · 8 min read
Pillar · molecular cloning
Plasmid cloning from insert to colony
How an insert becomes a plasmid, a colony and a verified clone, and which checkpoint fails when the plate is empty or the insert is wrong.
8 October 2026 · 8 min read
molecular cloning
Promoters tags and reading frames
How to check a promoter, a fusion tag and the reading frame on a plasmid map before you transform, including the ribosome binding site, stops and the junction.
8 October 2026 · 10 min read
molecular cloning
Restriction ligation and Gibson assembly
How restriction ligation and Gibson assembly join DNA ends, where each method fails, and what colony screening can and cannot confirm.
8 October 2026 · 8 min read
molecular cloning
Sanger confirmation of a clone
How to confirm a clone by Sanger sequencing: place the primer upstream, send clean DNA, plan a few hundred reliable bases, and read mixed peaks as mixed
8 October 2026 · 8 min read
molecular cloning
Synthetic DNA versus PCR-amplified inserts
Choose a synthetic fragment or a PCR copy by polymerase error, codon changes, and the checks both still need before the sequence is trusted.
8 October 2026 · 7 min read
molecular cloning
Troubleshooting lawns and satellites
Tell a lawn from ampicillin satellites, decide whether selection failed, and stop before you pick a colony that is only living in a cleared zone.
8 October 2026 · 8 min read
molecular cloning
Troubleshooting plates with no colonies
An empty plate is a place to define competent cells, ligation, selection, and transformation efficiency, then test the failure that actually fits.
8 October 2026 · 7 min read